Bioinformatics to Identify Biomarkers of Diabetic Nephropathy based on Sphingolipid Metabolism and their Molecular Mechanisms.

ACKGROUND: Diabetes mellitus (DM) frequently results in Diabetic Nephropathy (DN), which has a significant negative impact on the quality of life of diabetic patients. Sphingolipid metabolism is associated with diabetes, but its relationship with DN is unclear. Therefore, screening biomarkers related to sphingolipid metabolism is crucial for treating DN. METHODS: To identify Differentially Expressed Genes (DEGs) in the GSE142153 dataset, we conducted a differential expression analysis (DN samples versus control samples). The intersection genes were obtained by overlapping DEGs and Sphingolipid Metabolism-Related Genes (SMRGs). Furthermore, The Least Absolute Shrinkage and Selection Operator (LASSO) and Support Vector Machine Recursive Feature Elimination (SVM-RFE) algorithms were used to filter biomarkers. We further analyzed the Gene Set Enrichment analysis (GSEA) and the immunoinfiltrational analysis based on biomarkers. RESULTS: We identified 2,186 DEGs associated with DN. Then, five SMR-DEGs were obtained. Subsequently, biomarkers associated with sphingolipid metabolism (S1PR1 and SELL) were identified by applying machine learning and expression analysis. In addition, GSEA showed that these biomarkers were correlated with cytokine cytokine receptor interaction'. Significant variations in B cells, DCs, Tems, and Th2 cells between the two groups suggested that these cells might have a role in DN. CONCLUSION: Overall, we obtained two sphingolipid metabolism-related biomarkers (S1PR1 and SELL) associated with DN, which laid a theoretical foundation for treating DN.

Exosome miR-30a-5p Regulates Glomerular Endothelial Cells' EndMT and Angiogenesis by Modulating Notch1/VEGF Signaling Pathway.

BACKGROUND: Diabetic nephropathy (DN) is one of the microvascular complications of diabetes. Endothelial-mesenchymal transition (EndMT) and endothelial damage lead to abnormal angiogenesis in DN. OBJECTIVES: This study aimed to investigate the role of exosome miR-30a-5p in high glucose (HG)-induced glomerular endothelial cells (GECs) dysfunction and explore the underlying mechanisms. METHODS: GECs were cultured in normal glucose (5.5 mM) and HG (30 mM) conditions. The recipient GECs were transfected with exosome or miR-30a-5p mimic/inhibitor and then detected by using CCK-8 and flow cytometry assay. Luciferase analysis was used to verify miR-30a-5p acted on notch homolog protein 1 (Notch1). RT-qPCR and Western blot were used to detect the expression of VE-cadherin, α-SMA, vascular endothelial growth factor (VEGF) and Notch1. In vivo, exosome miR-30a-5p was administered to DN mice, and periodic acid-Schiff (PAS) staining, UTP levels, and HbA1c levels were measured. RESULTS: The expression of miR-30a-5p was downregulated in HG-treated GECs. Exosome miR-30a-5p significantly promoted cell proliferation, and migration and reduced apoptosis of GECs under HG conditions. MiR-30a-5p directly targeted the 3-UTR region of Notch1. Exosome miR-30a-5p reduced the expression levels of Notch1 and VEGF, both at mRNA and protein levels. Furthermore, exosome miR-30a-5p inhibited HG-induced EndMT, as evidenced by increased VE-cadherin and reduced α-SMA. In vivo studies demonstrated that exosome miR-30a-5p reduced serum HbA1c levels and 24-hour urine protein quantification. CONCLUSION: This study provides evidence that exosome miR-30a-5p suppresses EndMT and abnormal angiogenesis of GECs by modulating the Notch1/VEGF signaling pathway. These findings suggest that exosome miR-30a-5p could be a potential therapeutic strategy for the treatment of DN.

Evaluation of malnutrition and inflammation after total parathyroidectomy in patients on maintenance dialysis.

PURPOSE: To evaluate the effect of total parathyroidectomy (tPTx) on malnutrition and inflammation in patients on maintenance dialysis (MHD) having secondary hyperparathyroidism (SHPT). METHODS: Twenty-five patients on MHD having SHPT who were being treated with tPTx were selected, and changes in their general condition (dry body mass), parathyroid hormone (PTH) and calcium levels, nutrition state (hemoglobin, hematocrit, serum albumin, and total iron binding capacity), and inflammatory status [serum C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and the malnutrition-inflammation score (MIS)] were observed at 12, 24, and 36 months postoperatively. RESULTS: Compared with the preoperative period, the dry body mass increased at 12, 24, and 36 months postoperatively (P < 0.01), hemoglobin, hematocrit, and serum albumin increased significantly (P < 0.01), whereas calcium, phosphorus, and PTH levels decreased significantly (P < 0.01). Serum CRP, IL-6, and TNF-α levels were significantly decreased at 12, 24, and 36 months after surgery (P < 0.01). Furthermore, MIS was reduced as well but to a lesser extent (P < 0.01). CONCLUSION: tPTx effectively reduced MIS in maintenance dialysis patients, and the alleviated malnutrition and improved inflammatory status may contributed to improving the quality of life of patients on MHD with SHPT.

miR­152 regulates TGF­ß1­induced epithelial­mesenchymal transition by targeting HPIP in tubular epithelial cells.

Renal fibrosis is a common pathological feature of chronic kidney diseases, and their development and progression are influenced by epigenetic modifications including aberrant microRNA (miRNA or miR) expression. miRNAs have been demonstrated to modulate the aggressiveness of various cancers and have emerged as possible therapeutic agents for the management of renal fibrosis. Transforming growth factor ß1 (TGF­ß1)­induced epithelial­mesenchymal transition (EMT) of tubular epithelial cells serves a role in the initiation and progression of renal fibrosis. Furthermore, recent results indicated that the progression of EMT is reversible. The present study aimed to clarify the role of miR­152 in EMT of the tubular epithelial cell line HK­2, stimulated by TGF­ß1, using in vitro transfection with a miR­152 mimic and to further investigate the underlying mechanism of miR­152 activity. In the present study, miR­152 expression was significantly reduced in TGF­ß1­treated HK­2 cells, accompanied by an increased expression of hematopoietic pre­B­cell leukemia transcription factor (PBX)­interacting protein (HPIP). Additionally, miR­152 overexpression inhibited TGF­ß1­induced EMT and suppressed HPIP expression by directly targeting the 3' untranslated region of HPIP in HK­2 cells. Furthermore, upregulation of HPIP reversed miR­152­mediated inhibitory effects on the EMT. Collectively, the results suggest that downregulation of miR­152 initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which may provide important targets for prevention strategies of renal fibrosis.

[MiR-155 Inhibits the Expression of MMPs in HK2 Cells by Targeting KLF4].

OBJECTIVES: To determine whether miR -155 inhibits the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in HK2 cells by targeting Kruppel-like factor 4 (KLF4). METHODS: MiR -155-mimic, miR -155-NC empty plasmid, and culture medium were transfected into renal tubular epithelial cells, respectively. Six hours later, the expression of miR -155 was detected by real time-PCR; the expressions of MMP-2, MMP-9 and KLF4 were detected by Western blot; and the activity of MMP-2 and MMP-9 in the cells was detected by gelatin zymography. Because KLF4 was predicted as the target gene of miR -155 by bioinformatics. The miR-155 overexpressed HK2 cells were transfected with KLF4 overexpression plasmid or empty plasmid. Six hours later, the expressions of MMP-2, MMP-9 and KLF4, and the activity of MMP-2 and MMP-9 were measured again. Finally, cells containing luciferase plasmids with KLF4-3!d-UTR wild type (WT) or mutant (MUT) sequence were constructed and transfected with miR -155-mimic or empty plasmid. Luciferase assay was used to confirm whether KLF4 -3!d-UTR was the binding site in targeting miR -155#. RESULTS: Compared with the cells transfected with empty plasmid, the expression of miR -155 was up-regulated and the expressions of MMP-2, MMP-9 and KLF4 were down-regulated in the cells transfected with miR -155-mimic. Compared with the cells transfected with miR -155 mimic or miR -155 mimic+empty plasmid, the expressions of MMP-2, MMP-9 and KLF4 were up-regulated in the KLF4+miR -155 transfected cells. Luciferase assays confirmed that miR -155 binds to KLF4 , and KLF4 -3!d-UTR is the target gene of miR -155. CONCLUSION: MiR-155 inhibits the expressions of MMP-2 and MMP-9 in HK2 cells by targeting KLF4 -3!d-UTR.